The transformation of cultured cells with foreign DNA sequences is useful in the study of gene expression and in the production of commercially important heterologous gene products, such as valuable proteins. Simple proteins may be produced in bacterial cells. However, to function properly, many eukaryotic proteins require post-translational modifications that are not carried out by prokaryotic cells. There are other problems associated with expressing some proteins in prokaryotic cells; for example, some expressed heterologous proteins are deposited as insoluble inclusion bodies in prokaryotic cells, making the proteins difficult to recover. Many of the difficulties associated with prokaryotic expression systems may be overcome by using transformed mammalian cell culture systems to produce post-translationally processed proteins. Mammalian cell cultures may, however, be relatively inefficient because they grow slowly and are difficult and costly to maintain.
Advances in the culture of insect cells, and the development of baculovirus-based expression systems, have facilitated the expression of heterologous proteins by transformed insect cell lines (Luckow and Summers, Bio/Tech., 6: 47–55 (1988); Miller, Annu. Rev. Microbiol., 42: 177–199 (1988)). To date, the expression of heterologous proteins in transformed insect cell lines has been accomplished primarily using vectors derived from the baculovirus Autographa californica multicapsid nucleopolyhedrosis virus (AcMNPV) (Luckow and Summers, Bio/Tech., 6: 47–55 (1988); Miller, Annu. Rev. Microbiol., 42: 177–199 (1988)). Baculoviruses are double-stranded DNA viruses that kill infected insect cells by lysis at the end of a typical infection cycle. A variety of baculoviruses are known, each of which is endemic to a particular arthropod species. Baculoviruses are not known to undergo replication in animals outside the Arthropoda. An understanding of the prior art in this field requires some appreciation of the molecular biology of baculovirus infection.
Gene expression during natural baculovirus infection of an insect is highly regulated and occurs as an ordered cascade. The viral genes may be classified into four different groups according to their place in this cascade of gene expression: immediate early (ie), delayed early (de), late, and very late. Early gene expression occurs before the onset of viral DNA replication and appears to be essential for the induction of late viral gene expression (Blissard and Rohrmann, Annu. Rev. Entomol., 35: 127–155 (1990); Guarino and Summers, J. Virol., 62: 463–471(1988); Miller et al., Virology, 126: 376–380 (1983)). Experimental evidence indicates that baculovirus ie genes are transcribed by host RNA polymerase II in the absence of other viral factors. Baculovirus ie genes are therefore understood to have promoters that are recognized by the host cell transcription machinery.
In prior art expression systems based on derivatives of the AcMNPV, foreign gene expression is generally directed by a very strong late viral promoter, such as the polyhedrin (pol) or p10 promoters. Expression from such baculovirus late promoters is, however, dependent upon viral-encoded RNA polymerase for transcription and is restricted to permissive lepidopteran cells, ie. cells that permit lytic baculovirus infection (Carbonell et al., J. Virol., 56: 153–160 (1985)). A wide array of baculovirus expression vectors have been designed to optimize expression, secretion and recovery of recombinant proteins produced by such systems (O'Reilly et al., Baculovirus Expression Vectors, W. H. Freeman and Company, New York, N.Y., USA (1992); U.S. Pat. No. 5,179,007 to Jarvis and Carrington; Lenhard et al., Gene, 169: 187–190 (1996)). Many post-translational modifications known to occur in mammalian systems, including N- and O-linked glycosylation, phosphorylation, acylation, proteolysis (Kidd and Emery, Appl. Biochem. Biotechnol., 42: 137–159 (1993)) and amidation (Andersons et al., Biochem. J., 280: 219–224 (1991)) also occur, at least to some degree, in insect cell lines infected with derivatives of the AcMNPV.
Using A cMNPV-based expression systems, proteins localized to the nucleus or cytoplasm may be expressed in adequate quantities (U.S. Pat. No. 5,179,007 to Jarvis et al. issued 12 Jan. 1993). Proteins entering the secretory pathway associated with the endoplasmic reticulum are, however, often expressed at lower levels (Jarvis, Insect Cell Culture Engineering, Marcel Dekker, Inc, New York, N.Y., USA (1993)). This subset of highly modified, membrane-bound and secreted proteins includes important bioactive species such as cell surface receptors (Chazenbalk and Rapoport, J. Biol. Chem., 270: 1543–1549 (1995)), antibodies (Hsu et al., Prot. Expr. Purif, 5: 595–603 (1994)) and secreted vaccine components (Li et al., Virology, 204: 266–278 (1994)). Proteins of this kind are frequently expressed relatively poorly and in a heterogeneous form in lytic AcMNPV-based systems. Reduced expression levels and alterations in processing may be the result of damage to the infected cells normal protein expression machinery caused by the progression of the lytic baculovirus infection (Kretzchmar et al., J. Biol. Chem., 375: 323–327 (1994); Jarvis and Finn, Virology, 212: 500–511 (1995); Chazenbalk and Rapoport, J. Biol. Chem., 270: 1543–1549 (1995)). Accordingly, research has been directed toward the generation of baculovirus vectors capable of expressing proteins early in the infection cycle (Jarvis and Finn, Nature Biotechnology, 14: 1288–1292 (1996); Jarvis et al., Prot. Expr. Purif., 8: 191–203 (1996)).
To overcome the problems associated with lytic baculovirus expression systems, approaches have been developed for the stable transformation of insect cell lines. Drosophila melanogaster Schneider cells have been stably transformed with a system that utilizes the D. melanogaster metallothionein promoter to drive heterologous protein expression and hygromycin selection to identify transformants (Johansen et al., Genes Develop., 3: 882–889 (1989); Culp et al., Bio/Technology, 9: 173–177 (1991)). Dipteran cell lines (D. melanogaster and Aedes albopictus, mosquito) have been stably transformed with a system that utilizes the D. melanogaster hsp70 or AcMNPV ie1 promoters to drive heterologous protein expression and methotrexate selection to identify transformants (Shotkoski et al., FEBS Lett., 380: 257–262 (1996)). A lepidopteran cell line (Sf9, derived from the fall army worm Spodoptera frugiperda) has been stably transformed with a system that utilizes the AcMNPV ie1 promoter to drive heterologous protein expression and geneticin (G-418) selection to identify transformants, although expression in this system was found to be relatively inefficient (Jarvis et al., Bio/Technology, 8: 950–955 (1990); U.S. Pat. No. 5,077,214 issued to Guarino and Jarvis on 31 Dec. 1991). In each of these transformation systems, the selectable marker on one vector was cotransfected with a separate expression vector carrying the heterologous protein expression cassette. Using separate plasmids that must be cotransfected complicates the transformation procedure, since some of the cell lines that acquire the selectable marker will not also acquire the desired expression vector. There is accordingly a need in the art for vectors capable of providing both a selectable marker and an expression cassette.
There is also a need in the art for strong promoters to direct expression of heterologous proteins in stably transformed insect cells. In an attempt to meet this need, the hr enhancer element has been used to increase expression from the Ac ie1 promoter (Shotkoski et al., FEBS Lett., 380: 257–262 (1996)). The hr enhancer exists as five large homologous regions dispersed throughout the AcMNPV baculovirus genome and serve to activate transcription of proximal genes (Leisy et al., Virology, 208: 742–752 (1995)). However, difficulties may arise with the use of hr elements in transformation systems because specific cellular or baculovirus-encoded factors may be required to modulate the action of hr elements (Glocker et al., J. Virol., 66: 3476–3484 (1992); Choi and Guarino, J. Virol., 69: 4548–4551 (1995); Rodems and Friesen, J. Virol., 69: 5368–5375 (1995)). Also, adding enhancer sequences to a promoter may significantly increase the size of the promoter, necessarily leaving less room in the relevant vector for the heterologous gene of interest. There is therefore a need in the art for promoters that are capable of directing adequate levels of heterologous protein expression, including selectable marker expression, without the need for enhancer sequences.
Transposable elements have been used as transformation vectors in a number of organisms. Transposable elements are mobile segments of DNA that are characterized by the ability to autonomously replicate and insert themselves in a variety of locations within the cell's genome. There are two distinctly different classes of transposable elements: 1) the short inverted repeat class of DNA transposons (“DNA transposable elements”); and, 2) the retrotransposons which replicate through an RNA intermediate and require reverse transcriptase activity for transposition (such as are disclosed in International Patent Publication Number WO 88/03169). One aspect of the present invention relates to the short inverted repeat class of DNA transposable elements, as distinguished from retrotransposons.
A complete DNA transposable element encodes a transposase enzyme that mediates transposition of the element. The transposase protein interacts with DNA sequences near the termini of the element; intact termini (usually about 150 to 250 base pairs) are typically required to allow DNA transposable elements to respond to the transposase enzyme.
The DNA transposable elements, P, hobo, mariner, I, and Hermes (a hobo-like mobile element from Musca domestica) have all been used to transform the fruit fly, D. melanogaster (O'Brochta, et al., J. of Cell. Biochemistry-Keystone Symposia Suppl., 21A: 195 (1995); Pritchard, et al., Mol. Gen. Genet., 214: 533–540 (1988)). Large pieces of foreign DNA (>12 kb) can be placed within non-coding regions of the P element and not hinder its ability to replicate through transposition (Meister and Grigliatti, Genome, 36: 1169–1175 (1993)). The DNA transposable element Tc1 has been used to transform the round worm Caenorhabdites elegans. The selection of desired transformants is an important step in any transformation system. While several transformation systems based upon auxotrophic complementation or dominant selection have been designed for use in mammalian systems, relatively few have been adapted for insect cells (Walker, Adv. Cell Cult., 7: 87–124 (1989); Carlson et al., Annu. Rev. Entomol., 40: 359–388 (1995)). The transformation of D. melanogaster cells to methotrexate resistance using a bacterial dihydrofolate reductase (DHFR) gene was first described by Bourouis and Jarry, EMBO J., 2: 1099–1104 (1983). Subsequently, Shotkoski and Fallon, Insect Biochem. Molec. Biol., 23: 883–893 (1993) described a mosquito dihydrofolate reductase gene that functioned as a dominant selectable marker in mosquito cells. In these instances the transforming DNA was incorporated into the genome as repetitive structures and as randomly integrated single copies; however, in the absence of selective pressure a loss of transfecting DNA was observed (Shotkoski and Fallon, Insect Biochem. Molec. Biol., 23: 883–893 (1993)). Resistance to geneticin (G418) after introduction of the bacterial neomycin phosphotransferase gene, can be endowed upon both D. melanogaster (Steller and Pirotta, EMBO J., 4: 167–171 (1985)) and its derivative cell lines (Rio and Rubin, Mol. Cell. Biol., 5: 1833–1838 (1985)), mosquitoes (Maisonhaute and Echalier, FEBS Lett., 197: 45–49 (1986); Lycett and Crampton, Gene, 136: 129–136 (1993)) and the Sf9 lepidopteran cell line (Jarvis et al., Bio/Tech., 8: 950–955 (1990)). However, gene amplification arising from continued selection and high spontaneous resistance frequencies (McGrane et al., Am. J. Trop. Med Hyg., 39: 502–510 (1988) undermine the use of this selection system in certain instances. Hygromycin resistance provided by the bacterial hygromycin B phosphotransferase gene is reported to be more reliable, and selection more rapid, than in G418-based selection systems (van der Straten et al., Invertebrate Cell System Applications, CRC Press Inc., Boca Raton, Fla., USA (1989); Carlson et al., Annu. Rev. Entomol., 40: 359–388 (1995)). However, in mosquito cell lines transformed for hygromycin resistance, the introduced plasmid was amplified extensively and was present as long tandem arrays or as self-replicating extra chromosomal pseudo-chromosomes (Monroe et al., Proc. Natl. Acad. Sci. USA, 89: 5725–5729 (1992)). Either genetic arrangement, tandem arrays or extra chromosomal elements, lends itself to rapid loss of the resistance gene once selection has been relaxed. Accordingly, there is a need for an improved selection system and strategy for efficient insertion of DNA into the host cells' genome, particularly for use in selecting stably transformed insect cells.
Zeocin is a member of the bleomycin/phleomycin family of antibiotics isolated from Streptomyces verticillus (Berdy, Handbook of Antibiotic Compounds, Vol IV, Part 1. Amino Acid and Peptide Antibiotics, CRC Press, Boca Raton, Fla., USA (1980)). Zeocin is a trademark of S.A.R.L. Cayla of Toulouse, France, from whom it may be available. Zeocin is a copper-chelated glycopeptide of the formula C55H83N19O21S2Cu.
Resistance to the bleomycin/phleomycin family of antibiotics may be conferred by a 3.6 kDa protein, the product of the Streptoalloteichus hindustanus ble gene, that binds the antibiotic in a stoichiometric manner (Gatignol et al., FEBS Lett., 230: 171–175 (1988)). The ble resistance gene has been successfully used in mammalian (Mulsant et al., Somat. Cell Mol. Genet., 14: 243–252 (1988)) and plant cells (Perez et al., Plant Mol. Biol., 13:365–373 (1989)) to confer Zeocin resistance. The effect the bleomycin/phleomycin antibiotics on cells derived from other genera is unpredictable.
A number of potential difficulties are associated with the use of Zeocin as a selectable marker. The copper-chelated form of the drug is inactive. The current incomplete understanding of the mechanism of action of the Zeocin suggests that activation only occurs if appropriate conditions are encountered within the target cell to reduce the chelated copper from Cu2+ to Cu1+, so that the copper ion may be removed by sulfydryl compounds in the cell. High salt concentrations may inactivate Zeocin. The drug may also be inactivated by acidic or basic solutions (Invitrogen Corporation “pZeoSV2(+) or pZeoSV2(−)” product manual, Version C, San Diego, Calif., U.S.A.).
Insect cell expression systems are of interest in large part because of their ability to accomplish sophisticated post translational modifications. However, there may be variability from one cell line to another in the nature of the precise post translational modification to a protein of interest. Accordingly, it may be useful to screen a number of transformed insect cell lines from disparate species to determine which cell line best expresses the protein of interest. To accomplish such a screening procedure, there is a need in the art for an expression vector capable of stably transforming a range of insect cell lines to strongly express heterologous proteins.